A short proximal promoter and the distal hepatic control region-1 (HCR-1) contribute to the liver specificity of the human apolipoprotein C-II gene. Hepatic enhancement by HCR-1 requires two proximal hormone response elements which have different binding specificities for orphan receptors HNF-4, ARP-1, and EAR-2.
We have identified the regulatory elements, some of the factors and potential regulatory mechanisms which determine the tissue specificity of the human apoC-II gene. The -545/+18 apoC-II promoter directs high levels of expression of the reporter CAT gene in cells of hepatic origin (HepG2), low levels of expression in cells of intestinal origin (CaCo-2) and basal expression in HeLa cells. Deletion analysis identified negative regulatory elements within the -545/-388 region and positive regulatory elements within the -388/-55 region. Linkage of different apoC-II promoter segments to the hepatic control region-1 (HCR-1) enhanced the promoter activity 2.5-11-fold in HepG2 cells but did not affect its activity in CaCo-2 or COS-1 cells. DNase I footprinting analysis using rat liver nuclear extracts identified five protected regions within the -545/+18 apoC-II promoter as follows: CIIA (-74/-44), CIIB (-102/-81), CIIC (-159/-116), CIID (-288/-265), and CIIE (-497/-462). Elements CIIB and CIIC contain hormone response elements. CIIB is recognized by hepatic nuclear factor-4 (HNF-4) but not ARP-1 or EAR-2, whereas CIIC is recognized by ARP-1 and EAR-2 but not by HNF-4. HNF-4 transactivated the apoC-II promoter or the apoC-II promoter linked to the HCR-1 in COS-1 cells. A double mutation in elements CIIB and CIIC that eliminated binding of HNF-4 or ARP-1 and EAR-2, respectively, to these sites abolished the enhancer activity of HCR-1. The combined data suggest that the apoC-II promoter/HCR-1 cluster can direct expression in cells of hepatic origin and that optimal enhancer activity requires synergistic interactions between factors bound to the distal HCR-1 and nuclear receptors bound to the two proximal hormone response elements.
Vorgia
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Division of Basic Sciences, Section of Biochemistry, Department of Medicine, University of Crete and the Institute of Molecular Biology and Biotechnology, Herakleion 71110, Crete, Greece.
Zannis
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Kardassis
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D000818:Animals; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002478:Cells, Cultured; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000032:analysis; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008099:Liver / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics
D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D014157:Transcription Factors